Twenty-six mutant forms of simian immunodeficiency virus train mac239 were constructed with changes in constant region 4 (C4) of env. Twenty-four of these had a single amino acid change, one had changes in two amino acids, and one had a deletion of eight amino acids. The effects of these mutations on viral replication, gp160 procession, and binding of env protein to soluble CD4 receptor were analyzed. The C4 region was relatively sensitive to sequence changes since only 11 of the 26 mutants replicated appreciably. Eight of the 15 mutants that were replication incompetent exhibited grossly defective processing of the gp160 env precursor; these mutations likely resulted in global effects on gp160 structure. Six of the replication incompetent mutants exhibited normal or near normal gp160 processing and binding of env protein to sCD4 and thus were probably blocked at some step subsequent to binding of virus to its CD4 receptor. Only one of the C4 mutations, 441W R, resulted in greatly decreased binding to sCD4 while retaining normal processing of gp160. The equivalent residue in HIV-1 has similarly been shown previously to be important for binding of HIV-1 to the CD4 receptor. Since a W S mutation at position 441 in C4 of SIVmac239 affected both gp160 processing and sCD4 binding, it is not clear whether the 441 tryptophan is actually important for contacting CD4 or for maintaining an appropriate configuration. Mutations within a highly conserved GGDPE sequence in C3 of SIVmac239 specifically affected CD4 binding, which is also similar to previous findings with HIV-1. These results demonstrate similar sequence requirements in SIVmac and HIV-1 env for binding CD4, but they raise doubts as to whether C4 sequences are directly involved in the binding.